TRPC1 and TRPC5 form a novel cation channel in mammalian brain.

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.

A switch mechanism for G beta gamma activation of I(KACh).

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.

pICln inhibits snRNP biogenesis by binding core spliceosomal proteins.

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.

pICln binds to a mammalian homolog of a yeast protein involved in regulation of cell morphology.

Since its cloning and tentative identification as a chloride channel, the function of the pICln protein has been debated. Although there is no consensus regarding the specific function of pICln, it was suggested to play a role, directly or indirectly, in the function of a swelling-induced chloride conductance. Previously, the protein was shown to exist in several discrete protein complexes. To determine the function of the protein, we have begun the systematic identification of all proteins to which it binds. Here we show that four proteins firmly bind to pICln and identify the 72-kDa pICln-binding protein by affinity purification and peptide microsequencing. The interaction between this protein and pICln was verified several ways, including the extraction of several pICln clones from a cDNA library using the 72-kDa protein as a bait in a yeast two-hybrid screen. The protein is homologous to the yeast Skb1 protein. Skb1 interacts with Shk1, a homolog of the p21(Cdc42/Rac)-activated protein kinases (PAKs). The known involvement of PAKs in cytoskeletal rearrangement suggests that pICln may be linked to a system regulating cell morphology.

Gbeta binding to GIRK4 subunit is critical for G protein-gated K+ channel activation.

The cardiac G protein-gated K+ channel, IKACh, is directly activated by G protein beta gamma subunits (Gbeta gamma). IKAChis composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. Gbeta gamma binds to both GIRK1 and GIRK4 subunits of the heteromultimeric IKACh. Here we delineate the Gbeta gamma binding regions of IKACh by studying direct Gbeta gamma interaction with native purified IKACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in Gbeta gamma binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for Gbeta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting Gbeta gamma binding and Gbeta gamma activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of Gbeta gamma binding to IKACh and Gbeta gamma activation of GIRK1/mutant GIRK4 channels. We conclude from this data that Gbeta gamma binding to GIRK4 is critical for IKACh activation.

A novel inward rectifier K+ channel with unique pore properties.

We have cloned a novel K+-selective, inward rectifier channel that is widely expressed in brain but is especially abundant in the Purkinje cell layer of the cerebellum and pyramidal cells of the hippocampus. It is also present in a wide array of tissues, including kidney and intestine. The channel is only 38% identical to its closest relative, Kir1.3 (Kir1-ATP-regulated inward rectifier K+ [ROMK] family) and displays none of the functional properties unique to the ROMK class. Kir7.1 has several unique features, including a very low estimated single channel conductance (approximately 50 fS), low sensitivity to block by external Ba2+ and Cs+, and no dependence of its inward rectification properties on the internal blocking particle Mg2+. The unusual pore properties of Kir7.1 seem to be explained by amino acids in the pore sequence that differ from corresponding conserved residues in all other Kir channel proteins. Replacement of one of these amino acids (Met-125) with the Arg absolutely conserved in all other Kir channels dramatically increases its single channel conductance and Ba2+ sensitivity. This channel would provide a steady background K+ current to help set the membrane potential in cells in which it is expressed. We propose that the novel channel be assigned to a new Kir subfamily, Kir7.1.

Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh.

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.

The G protein beta gamma subunit transduces the muscarinic receptor signal for Ca2+ release in Xenopus oocytes.

At least 30 G protein-linked receptors stimulate phosphatidylinositol 4,5-bisphosphate phosphodiesterase (phospholipase C beta, PLC beta) through G protein subunits to release intracellular calcium from the endoplasmic reticulum (Clapham, D. E. (1995) Cell 80, 259-268). Although both G alpha and G beta gamma G protein subunits have been shown to activate purified PLC beta in vitro, G alpha q has been presumed to mediate the pertussis toxin-insensitive response in vivo. In this study, we show that G beta gamma plays a dominant role in muscarinic-mediated activation of PLC beta by employing the Xenopus oocyte expression system. Antisense nucleotides and antibodies to G alpha q/11 blocked the m3-mediated signal transduction by inhibiting interaction of the muscarinic receptor with the G protein. Agents that specifically bound free G beta gamma subunits (G alpha-GDP and a beta-adrenergic receptor kinase fragment) inhibited acetylcholine-induced signal transduction to PLC beta, and injection of G beta gamma subunits into oocytes directly induced release of intracellular Ca2+. We conclude that receptor coupling specificity of the G alpha q/G beta gamma heterotrimer is determined by G alpha q; G beta gamma is the predominant signaling molecule activating oocyte PLC beta.

The cardiac inward rectifier K+ channel subunit, CIR, does not comprise the ATP-sensitive K+ channel, IKATP.

Cardiac IKACh is comprised of two inwardly rectifying K+ channel subunits, CIR and GIRK1 (Krapivinsky, G., Gordon, E. G., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). A cardiac protein virtually identical to CIR, termed rcKATP-1 (Ashford, M. L. J., Bond, C. T., Blair, T. A., and Adelman, J. P. (1994) Nature 370, 456-459), was reported to form an ATP-sensitive inwardly rectifying K+ channel, IKATP. We attempted to determine whether CIR alone or together with an unknown protein(s) participated in the formation of cardiac IKATP. Expression of CIR in insect, oocyte, and mammalian cell systems did not increase the appearance of ATP-sensitive currents, but rather gave rise to unique strongly inwardly rectifying, G protein-regulated K+ currents. CIR protein is found exclusively in atria, in contrast to the predominance of IKATP functional activity in ventricle. Also, CIR was completely depleted from heart membrane after immunodepletion of GIRK1. We conclude that CIR/rcKATP-1 is not a subunit of cardiac IKATP and that GIRK1 is the only channel protein coassociating with CIR in heart.

G beta gamma binds directly to the G protein-gated K+ channel, IKACh.

The cardiac G protein-gated K+ channel, IKACh, is activated by application of purified and recombinant beta and gamma subunits (G beta gamma) of heterotrimeric G proteins to excised inside-out patches from atrial membranes (Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E. (1987) Nature 325, 321-326; Wickman, K., Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E., Gilman, A., and Clapham, D.E. (1994) Nature 368, 255-257). Cardiac IKACh is composed of two inward rectifier K+ channel subunits, GIRK1 and CIR (Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We show that G beta gamma directly binds to immunoprecipitated cardiac IKACh as well as to recombinant CIR and GIRK1 subunits, with dissociation constants (Kd) of 55, 50, and 125 nM, respectively. In each case, binding appeared specific as judged by competition of unlabeled G beta gamma with radiolabeled G beta gamma and inhibition of binding by antigenic peptide or G alpha-GDP, but not G alpha-GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). In contrast, G alpha (GTP gamma S- or GDP-bound) did not bind to the native channel. We conclude that G beta gamma binds directly and specifically to IKACh via interactions with both CIR and GIRK1 subunits to gate the channel.

The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins.

Heart rate is slowed in part by acetylcholine-dependent activation of a cardiac potassium (K+) channel, IKACh. Activated muscarinic receptors stimulate IKACh via the G-protein beta gamma-subunits. It has been assumed that the inwardly rectifying K(+)-channel gene, GIRK1, alone encodes IKACh. It is now shown that IKACh is a heteromultimer of two distinct inwardly rectifying K(+)-channel subunits, GIRK1 and a newly cloned member of the family, CIR.

Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln.

Cells maintain control of their volume by the passage of KCl and water across their membranes, but the regulatory proteins are unknown. Expression in Xenopus oocytes of a novel protein, pICln, activated a chloride conductance. We have cloned analogs of pICln from rat heart and Xenopus ovary. pICln was identified as an abundant soluble cytosolic protein (approximately 40 kd) that does not immunolocalize with the plasma membrane. pICln was found in epithelial and cardiac cells, brain, and Xenopus oocytes, forming complexes with soluble actin and other cytosolic proteins. Monoclonal antibodies recognizing pICln blocked activation of a native hypotonicity-induced chloride conductance (ICl.swell) in Xenopus oocytes, suggesting that pICln may link actin-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. The high degree of sequence conservation and widespread expression of pICln suggest that it is an important element in cellular volume regulation.

Characterization of a native swelling-induced chloride current, ICl.swell, and its regulatory protein, pICln, in Xenopus oocytes.

The ability to precisely regulate cell volume is a fundamental property of most cells. Although the phenomenon of regulatory volume decrease (RVD), whereby a swollen cell loses salt and water to restore its original volume, has been appreciated for decades, the molecular identities of the proteins responsible for the volume control machinery and their regulation are essentially unknown. It appears that the rate-determining step in gaining volume control involves the activation of potassium and chloride conductance pathways. We have identified a native chloride current (ICl.swell) responsive to cell swelling in Xenopus oocytes [Ackerman et al. (1994) J Gen Physiol 103: 153-179]. Moreover, we have demonstrated that a cloned protein, pICln, endogenous to oocytes is critical for the activation of this volume-sensitive chloride conductance pathway [Krapivinsky et al. (1994) Cell 76: 439-448]. The identification of an endogenous protein participating in the regulation of an endogenous current may help understand the physiological activities of swelling-induced chloride channels.